Astrocytes in the human brain are initiated by elutriation from the cortical tissues of the dissociated normal human brain. HBA is shipped in proliferation culture or frozen vial with> 90% confluence (cells are provided in passage 1). Astrocyte Growth Medium (CAT # AGPM-03) containing 5% fetal bovine serum and a growth supplement for culture is recommended. Cells have an average population doubling level> 10 when cultured. When you receive the cells, leave the flask in the CO2 incubator at 37 ° C for 1 hour. Then replace the transport medium with a new Astrocyte Growth medium (CAT # AGPM-03). Allow cells to grow for 24 hours before subculturing.
If cells arrive frozen:
When you receive the cells in a frozen vial, you can transfer the cell vial at -80 ° C freezer for short-term storage or a liquid nitrogen tank for long-term storage.
- Coating of T25 flasks. Add 2 ml of AlphaBioCoat (CAT # AC001) to a T25 flask and make sure the entire interior surface is coated with the solution. After 30 minutes, discard AlphaBioCoat (CAT # AC001) by aspiration. Rinse and gently aspirate the flask with Phosphate buffer solution (1XPBS-001). The flask is now ready for use (no need to incubation overnight when coated with CAT # AC001)
- If you are using the coated flask on the same day, add approximately 4 ml of Astrocyte-Growth medium (CAT # AGPM-03) to the coated flask. * If the material changes from pink to yellow, vacuum and discard the medium. Add 4 ml of fresh medium to the coated flask.
- Thaw the cells in a 37 ° C water bath. Once you see a small amount of ice in the vial, spray the vial with 70% ethanol and clean it.
- Transfer the vial to your biosafety cabinet.
- Using a 2 or 5 ml pipet, remove the cells from the vial with a pipette.
- Transfer your cell suspension to your coated plate containing 4 ml medium.
- You must have a total working volume of 5 ml of cell suspension in the flask; close the cap. Make sure the cells are evenly distributed in the flask by moving the flask left and right five times. Move it up and down five more times.
- Place the flask in a 37 ° C incubator with 5% CO2. If the flask is not vented, loosen the cap.
- Change media after 48 hours.
If the cells arrive in a T25 flask:
Coating of T25 flasks. Add 2 ml of AlphaBioCoat (CAT # AC001) to 2 T25 flasks and make sure the entire interior surface is covered with the solution. After 30 minutes, discard AlphaBioCoat (CAT # AC001) by aspiration. Rinse and gently aspirate the flask with Phosphate buffer solution (1XPBS-001). The flask is now ready for use (no need to
incubation overnight when coated with CAT # AC001). Add new media to the flask, if the color change from pink to yellow, discard the medium and add a new medium to each flask.
- Inspect to make sure the flask is 90% confluent, if not, remove the transport medium and add 5 ml of fresh medium to the flask. Place the flask in a 37 ° C incubator until the cells are 90% confluence. Change the media every 2 days.
- If the flask is 90% confluent, aspirate the transport medium from the flask.
- Rinse the T25 flask containing the cells with 5 ml of 1XPBS-001
- Gently vacuum 1XPBS-001 after rinsing and discard.
- Add 2 ml of AlphabioDetach Cell Detachment Solution to the T25 flask (CAT # ADF001) containing cells (be sure to cover the entire inner surface).
- Place the T25 flask containing the cells in a 37 ° C incubator for 1 to 2 minutes (cells usually peel off the surface in 1 to 2 minutes). Monitor cell shedding. Strike the vial against the palm of the hand to separate the cells
- Suspend cells with 10 ml of astrocyte growth medium (CAT # AGPM-03) and transfer equally to 2 precoated T25 flasks (cells are now at a subculture ratio of 1: 2.
- It is not necessary to rotate cells during subculture.
- Culture of proliferating cells: Astrocyte growth medium (CAT # AGPM-03) must change every 2 days. Cells normally converge within 7 days (when they divide into a ratio of 1: 2)
- Use Astrocyte-Basal medium (CAT # AGPM-04) containing 0.5% FBS to induce inactivity.
cells (after 18-24 hours).
Format: Growing culture or frozen vial
Cell number: > 90% confluence in T25 /> 5×105 flask Frozen vial
Cytoplasmic GFAP: > 98% positive by immunofluorescence
Unit size: each
Suitability: Human brain astrocyte cells are for research use only
Safety statements: Handling human-derived products has the potential to be biologically dangerous. All Cell strains were negative for HIV, HBV, and HCV DNA in diagnostic tests. Adequate precautions must be taken to avoid exposure. Always wear the proper protective equipment (gloves, safety glasses, etc.) when handling these materials. We recommend following universal procedures for handling products of human origin as a minimum precaution against contamination.
Sample availability: No